Microfluidic Production of Uniform Microcarriers with

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Microfluidic Production of Uniform Microcarriers with Multicompartments through Phase Separation in Emulsion Drops Nam Gi Min, Minhee Ku, Jaemoon Yang, and Shin-Hyun Kim Chem. Mater., Just Accepted Manuscript • DOI: 10.1021/acs.chemmater.5b04798 • Publication Date (Web): 17 Feb 2016 Downloaded from http://pubs.acs.org on February 23, 2016

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Chemistry of Materials

Microfluidic Production of Uniform Microcarriers with Multicompartments through Phase Separation in Emulsion Drops Nam Gi Min,† Minhee Ku,‡, § Jaemoon Yang,‡, § and Shin-Hyun Kim*,† †

Department of Chemical and Biomolecular Engineering, KAIST, Daejeon 34141, South Korea

‡

Department of Radiology, College of Medicine, Yonsei University, Seoul 03722, South Korea

§

Brain Korea 21 plus Project for Medical Science, Yonsei University, Seoul 03722, South Korea

ABSTRACT: Microfluidics has provided biodegradable microcarriers for sustained release of drugs, while allowing insignificant initial bursting. Here, we further engineer the microcarriers to have multiple compartments to release distinct drugs in a more controlled fashion. With a capillary microfluidic device, monodisperse emulsion drops containing two immiscible biocompatible polymers—one is biodegradable and the other is pH-responsive—are prepared, which undergo phase separation as the solvent in the drops is depleted. The emulsion drops finally form uniform microcarriers with distinct compartments. During the phase separation and consolidation, two model drugs with different hydrophobicity involved in the drops are spontaneously concentrated in their own compartments with higher affinity. The configuration of the microcarriers is exclusively selected from five different structures depending on pair of polymers, pH of continuous phase, and organic solvent of emulsion drops; two of five have minimum interfacial energy and the others are kinetically arrested. Microcarriers with each configuration provide their unique release behavior of the model drugs. These are potentially useful for choosing release profile of multiple drugs proper to specific diseases.

INTRODUCTION Programmed release of multiple ingredients or drugs at controlled rate is of significant importance to improve therapeutic efficacy.1-5 Various types of drug carriers have been designed to fulfill the release functions.6-9 For example, uniform biodegradable microcarriers with spherical or cylindrical shapes are prepared by drop-based microfluidics and micromolding technique, respectively, to provide controlled rate of drug release with minimum initial bursting;10,11 conventional emulsion templates, prepared by bulk emulsification, yield polydisperse microspheres, achieving limited control over rate of polymer degradation and drug release.12 To encapsulate multiple distinct drugs and release them in programmed fashion, complex microcarriers such as Janus microparticles,13 capsules-in-capsules,14-16 and multi-cored capsules17-19 have been prepared. However, to produce such complex carriers, delicate flow control is usually required to produce complex emulsion templates and only small variation of carrier configuration is possible, thereby providing limited types of release profile; these restrict practical usage of the carriers. Although facile methods to create complex emulsions from single emulsions have been recently developed by employing controlled phase separation,20,21 it is still challenging to produce biocompatible, multicompartment microcarriers from the emulsions due to limited set of materials available for the liquid-liquid phase separation. Alternatively, microdisks and rods with multiple compartments have been designed by stop-flow lithography and sequential micromolding technique, respective-

ly.22,23 However, core-shell geometry that is good for encapsulation and controlled release is difficult to achieve using the techniques. A simple and versatile way to create multiple domains in single carrier is evaporation-induced phase separation of polymers in emulsion drops.24-28 For example, organic solution of two different biodegradable polymers is microfluidically emulsified in water, which form uniform microcarriers with dual domains after drop consolidation.29-31 However, resulting microcarriers show only few typical configurations with poorly controlled morphology. In addition, carrier materials are restricted to biodegradable polymers which are only appealing for sustained release, restricting applicable area. More importantly, encapsulation of distinct drugs in their own domains and their release behavior are yet to be exploited. In this paper, we report a pragmatic microfluidic method to create monodisperse microcarriers with multiple domains which are able to encapsulate two different materials in their own domains and to release them in a controlled fashion. With a capillary microfluidic device, organic solution containing biodegradable polymer and pHresponsive polymer is emulsified in water to form highly monodisperse emulsion drops. Drops evolve to microcarriers with multiple domains through phase separation as the organic solvent becomes depleted. During the phase separation, chemicals included in the drop spontaneously migrate into domains with higher affinity. Therefore, two different chemicals can be separately concentrated in their own domains. The configuration of the microcarriers is exclusively selected from several different shapes,

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including core-shell, acorn, inverted core-shell, coredouble shells, and multicores-in-matrix, according to a pair of polymers, organic solvent, and pH of collection liquid. The carrier configuration considerably influences the release behavior of encapsulants, which potentially enables us to select proper release profile of drugs to specific diseases.

RESULTS AND DISCUSSION Phase Separation of Polymers Confined in Emulsion Drops. When two immiscible polymers are diluted in the drop, they retain a single phase because random distribution of the polymers provides sufficient entropic energy gain. As the polymers are concentrated by solvent evaporation, disfavored interaction between two immiscible polymers overwhelms the entropic effects, resulting in spontaneous phase separation to minimize Gibbs free energy of mixing, Δ‫ܩ‬୫ ; Δ‫ܩ‬୫ has two minima and two phases have compositions corresponding to the minima. The phase separation is initiated by spinodal decomposition by which any small fluctuation in composition is amplified by diffusion inside the spinodal region.32 As a result, the small domains of one polymer-rich phase are surrounded by domains of the other polymer-rich phase, which are then coalesced and rearranged through Ostwald ripening to reduce interfacial energy.33 Finally, two large domains survive in each drop if they have sufficient fluidity. Two immiscible fluids dispersed in third immiscible continuous phase take one of four typical configurations at equilibrium to minimize total interfacial energy;34-40 the configurations include two drop-in-drop structures with opposite compositions, drop-to-drop linkage, and two drops isolated. The two domains in the drop also take similar configurations if there is no interruption toward equilibrium state. However, the solvent evaporation involves dynamic change of physical properties including interfacial energy and viscosity, which potentially yields rich variety of structural configurations that consolidated polymeric microcarriers eventually take. To provide various structures of microcarriers and achieve distinct release profiles for two different drugs, we employ the phase separation of two immiscible polymers within emulsion drops. One of the polymers is biodegradable, selected from poly(lactic-co-glycolic acid) (PLGA), polycaprolactone (PCL), and poly(lactic acid) (PLA) for sustained release and the other is poly(butyl methacrylate-co-(2-dimethylaminoethyl) methacrylateco-methyl methacrylate) (p(BMA-co-DAMA-co-MMA)) with 1:2:1 molar ratio, dissolvable in acidic condition, used for pH-responsive release. The polymers are dissolved in organic solvent of either chloroform or toluene. The binary polymer solution is uniformly emulsified into continuous phase using a glass capillary microfluidic device as shown in Figure S1 of the Supporting Information. As a continuous phase, we use aqueous solution of 10 w/w% polyvinyl alcohol (PVA) and 0.1 M NaOH. The solution has pH of 12 as prepared, which decreases over time to 8.5 within 3 h, as shown in Figure S2 of the Supporting

Figure 1. Schematic illustration showing the formation of microcarriers with five distinct configurations through phase separation between biodegradable and pH-responsive polymers confined in emulsion drops.

Information. This is because PVA is hydrolyzed in the basic condition which releases protons and neutralizes the solution. To control pH to be approximately 8.5 and 10 during phase separation in drops respectively, we collect drops for a short period of time and observe them; 5 minutes immediately after the first drop generation for pH 10 and 10 minutes after 1 h from the first drop generation for pH 8.5. Two fluorescent dyes of Nile red and Coumarin are additionally dissolved in the emulsion drops as model drugs. The emulsion drops evolve to monodisperse microcarriers, of which unique structure is exclusively selected from five distinct configurations as illustrated in Figure 1; three of them are difficult to create in absence of kinetic effect. The selection is influenced by composition of polymers, pH value of continuous phase, and organic solvent of emulsion drops. Microcarriers with Biodegradable Core and pHResponsive Shell. The emulsion drops of chloroform solution of hydrophobic PLGA and hydrophilic p(BMAco-DAMA-co-MMA) are homogeneous as prepared at approximately pH 8.5, which then become heterogeneous as they are shrunken by chloroform diffusion to the surrounding. Within drops, very small domains of p(BMAco-DAMA-co-MMA)-rich phases are formed in a continuous PLGA-rich phase by spinodal decomposition, which are continuously coalesced to form a large core in each drop. When the core is then brought into a contact with continuous phase, the core and shell phases are spontaneously inverted, finally resulting in the microcarriers composed of the PLGA-rich core and the p(BMA-coDAMA-co-MMA)-rich shell, as shown in Figure 2a, b and Movie S1 of the Supporting Information. The phase inversion is driven by interfacial energy. Because the interfacial tension between the p(BMA-co-DAMA-co-MMA)-rich and continuous phases is much lower than that between PLGA-rich and continuous phases, the p(BMA-co-DAMAco-MMA)-rich phase spreads on the surface of the PLGArich phase to minimize the interfacial area between the PLGA-rich and continuous phase; that is, spreading


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Chemistry of Materials contrast, hydrophobic Nile red has smaller interaction parameter with PLGA than p(BMA-co-DAMA-co-MMA), resulting in concentration in PLGA-rich phase.21 Therefore, drug molecules with different hydrophobicity can be also potentially encapsulated into their own domains. The core-shell microcarriers are stable against dissolution in neutral pH condition, thereby retaining the dyes. When the microcarriers are exposed to acidic condition of pH 5, the shell of p(BMA-co-DAMA-co-MMA) becomes selectively dissolved within 10 s, releasing the green dye to the surrounding as shown in Figure 2e and Movie S2 of the Supporting Information; release by pH- or specific enzyme-responsive dissolution of one compartment in Janus microparticles has been reported.13,40 Tertiary amines of DAMA groups in the polymer are ionized to be cationic in the acidic condition, being soluble in water. Biodegradable cores remain undissolved as shown in Figure S3 of the Supporting Information; in general, PLGA microsphere degrades in a time scale of a month.41

Figure 2. (a, b) Series of illustration and optical microscope (OM) images showing the formation of microcarrier composed of biodegradable core and pH-responsive shell from homogeneous chloroform drop containing p(BMA-coDAMA-co-MMA) and PLGA in aqueous phase with pH 8.5. Two different dye molecules of Nile red and Coumarin are additionally dissolved in the drop. (c) Confocal laser scanning microscope (CLSM) images of monodisperse core-shell microcarriers. (d) A set of red (left panel), green (middle panel), and overlaid (right panel) fluorescence CLSM images and intensity profile of red and green fluorescence along the dotted line in overlaid image. (e) A Series of CLSM images showing selective dissolution of pH-responsive shell at pH 5.

parameter for p(BMA-co-DAMA-co-MMA)-rich phase is positive.36,37 The core-shell geometry is maintained until drops are fully consolidated, yielding spherical core-shell microcarriers, as shown in Figure 2c and the inset of Figure 2c. The microcarriers are modeled by monodisperse emulsion drops, thereby showing narrow size distribution; average diameter is 25.70 μm and its coefficient of variation (CV) is as small as 1.58%. During the phase separation and consolidation, Nile red and Coumarin are spontaneously concentrated into PLGA-rich and p(BMA-coDAMA-co-MMA)-rich domains, respectively. Concentration of Nile red in the PLGA-rich core is approximately 2.23 times (CV: 12.6%) higher than that in the p(BMA-coDAMA-co-MMA)-rich shell, whereas concentration of Coumarin in the shell is approximately 1.79 times (CV: 20.5%) higher than that in the core, as shown in Figure 2d. This spontaneous, partial separation is attributed to opposite molecular affinity of dyes to polymers. Coumarin is relatively hydrophilic and has higher affinity to p(BMAco-DAMA-co-MMA) than PLGA; that is, Coumarin has smaller interaction parameter with p(BMA-co-DAMA-coMMA) than PLGA, which results in spontaneous migration of Coumarin toward p(BMA-co-DAMA-co-MMA)rich domains to minimize Gibbs free energy of mixing. By

Acorn-Shaped Janus Microcarriers. The chloroform drops with the same composition, PLGA and p(BMA-coDAMA-co-MMA), dispersed in the continuous phase with pH 10 evolve to acorn-shaped Janus microcarriers rather than core-shell. Drops experience the spinodal decomposition, subsequent internal coalescence, and phase inversion until the middle stage of phase separation in the same manner to continuous phase with pH 8.5. However, the p(BMA-co-DAMA-co-MMA)-rich phase dewets on the surface of the PLGA-rich phase, as shown in Figure 3a, b. The boundary between two polymeric phases is convex for the PLGA-rich core in the beginning of the dewetting of the shell, which become flat and then concave during the consolidation, ending up with acorn-shaped Janus microcarriers composed of p(BMA-co-DAMA-co-MMA)rich nut and PLGA-rich cupule, as shown in Figure 3c and the inset of Figure 3c. Nile red and Coumarin are concentrated into PLGA-rich and p(BMA-co-DAMA-co-MMA)rich phases, respectively in the same manner. Concentration of Nile red in the PLGA-rich cupule is approximately 3.15 times (CV: 12.2%) higher than that in the p(BMA-coDAMA-co-MMA)-rich nut, whereas concentration of Coumarin in the nut is approximately 2.72 times (CV: 21.4%) higher than that in the cupule, as shown in Figure 3d. This evolution from the core-shell structure to the acorn-shaped structure with PLGA-rich cupule is attributed to large interfacial tension between p(BMA-coDAMA-co-MMA)-rich and continuous phases at pH 10. DAMA groups in p(BMA-co-DAMA-co-MMA) are partially ionized at pH 8.5 which yields very small interfacial tension with the continuous phase, thereby leading to positive spreading parameter and complete core-shell structure. By contrast, the ionization is highly suppressed at pH 10 which increases the tension. As a result, spreading parameter becomes negative, leading to dewetting of the shell on the core.29-31 Moreover, the tension becomes even larger than that between the PLGA-rich and continuous phases, producing the acorn-shaped Janus microcarriers with PLGA-rich cupule. The acorn-shaped Janus



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Figure 3. (a, b) Series of illustration and OM images showing the formation of acorn-shaped microcarrier composed of pHresponsive nut and biodegradable cupule from homogeneous chloroform drop containing p(BMA-co-DAMA-co-MMA) and PLGA in aqueous phase with pH 10. (c) CLSM images of acorn-shaped microcarriers with uniform size and shape. (d) A set of red (left panel), green (middle panel), and overlaid (right panel) fluorescence CLSM images and intensity profile of red and green fluorescence along the dotted line in overlaid image. (e) A Series of CLSM images showing selective dissolution of pH-responsive nut at pH 5.

microcarriers also show selective dissolution of p(BMAco-DAMA-co-MMA)-rich domain and release of Coumarin when they are subjected to acidic condition as shown in Figure 3e and Movie S3 of the Supporting Information. The release is relatively anisotropic due to the geometry of microcarriers. After the dissolution, PLGA-rich domains with crescent-moon shape remain. Inverted Core-Shell Microcarriers. When PCL is used as biodegradable polymer, instead of PLGA, while maintaining the other conditions including solvent of chloroform, the final structures become phase-inverted core-shell at pH 8.5; core is p(BMA-co-DAMA-co-MMA)rich phase and shell is PCL-rich phase. Upon spinodal decomposition, small dispersed domains of p(BMA-coDAMA-co-MMA)-rich phase are formed in a similar manner to the combinations of PLGA and p(BMA-co-DAMAco-MMA). The domains coalesce to form large core and finally, microcarriers with p(BMA-co-DAMA-co-MMA)rich core and PCL-rich shell are prepared, as shown in Figure 4a-c; Coumarin and Nile red stain the core and shell, respectively. Concentration of Nile red in the PLGArich shell is approximately 1.86 times (CV: 2.37%) higher than that in the p(BMA-co-DAMA-co-MMA)-rich core, whereas concentration of Coumarin in the core is approximately 2.58 times (CV: 11.2%) higher than that in the

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Figure 4. (a, b) Series of illustration and OM images showing the formation of microcarrier composed of pH-responsive core and biodegradable shell from homogeneous chloroform drop containing p(BMA-co-DAMA-co-MMA) and PCL in aqueous phase with pH 8.5. (c) CLSM images of monodisperse inverted microcarriers. (d) A set of red (left panel), green (middle panel), and overlaid (right panel) fluorescence CLSM images and intensity profile of red and green fluorescence along the dotted line in overlaid image. (e) A Series of CLSM images showing very slow dissolution of pHresponsive core at pH 5.

shell, as shown in Figure 4d. We believe that inverted core-shell structures are thermodynamically unstable due to higher interfacial tension between PCL-rich and continuous phases than that between p(BMA-co-DAMA-coMMA)-rich and continuous phases; that is, spreading parameter for p(BMA-co-DAMA-co-MMA)-rich phase is positive. Nevertheless, phase separation leads to formation of p(BMA-co-DAMA-co-MMA)-rich dispersed phase in the PCL-rich surrounding, which are kinetically arrested by fast consolidation before experiencing the phase inversion; we observe the phase inversion at pH 10. The fast consolidation is attributed to low PVA coverage at interface. PVA becomes negatively charged by releasing protons to reduce pH from 12 to 8.5. Negatively charged PVA is unable to stabilize oil-water interface, possibly yielding low interfacial coverage. The inverted core-shell structures exhibit very slow dissolution of p(BMA-coDAMA-co-MMA)-rich core in acidic condition as shown in Figure 4e and Movie S4 of the Supporting Information. This is because the core is protected by biodegradable PCL shell. The intact shell allows limited diffusion of proton and prevents the release of Coumarin to surrounding. As the shell is partially degraded, the core releases Coumarin. After the release from the core, the shell persists for a longer period of time, slowly releasing Nile red. When the shell is incomplete due to formation of addi-


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tional small p(BMA-co-DAMA-co-MMA)-rich domains, the ionized polymer become released from the main core through the defects. The release is relatively fast in comparison with complete shell, but still slow in comparison with plain core-shell and acorn-shaped Janus microcarriers. The polymers become swollen in the core and then extruded through the defect, forming arms on the shell surface, as shown in Figure S4 of the Supporting Information. The release of the polymer leaves behind waterfilled core in the microcarrier. Microcarriers with Single Core and Double Shells. When emulsion drops are collected in a continuous phase with pH of 10, instead of pH of 8.5, while maintaining the other conditions including solvent of chloroform, the drops transform to microcarriers with single core and double shells; core and the outmost shell are p(BMA-coDAMA-co-MMA)-rich and inner shell is PCL-rich. In the early stage of phase separation, small p(BMA-co-DAMAco-MMA)-rich domains are formed in the PCL-rich surrounding, which then coalesce to form a large core in the same manner to pH 8.5. Afterward, the core-shell structures further undergo phase inversion before the phase separation is fully completed, forming p(BMA-co-DAMAco-MMA)-rich shell. Finally, remaining small p(BMO-coDAMA-co-MMA)-rich domains which do not participate in the phase inversion coalescence to form a single core as shown in Figure 5a, b. At pH 10, PVA is not fully ionized yet, being surface-active. Therefore, PVA strongly adsorbs at oil-water interface, which retards diffusion of chloroform. This provides a sufficient time for the phase inversion. After the phase inversion, the core further experiences phase separation and coalescence of p(BMA-coDAMA-co-MMA)-rich domains, eventually forming single p(BMA-co-DAMA-co-MMA)-rich core and double shells of PCL-rich inner layer and p(BMA-co-DAMA-co-MMA)rich outer layer as shown in Figure 5c, d; concentration of Nile red in the PLGA-inner shell is approximately 1.82 (CV: 8.05%) and 1.20 times (CV: 5.95%) higher than that in the p(BMA-co-DAMA-co-MMA)-rich outer shell and core, whereas concentrations of Coumarin in the outer shell and core are approximately 1.68 (CV: 10.7%) and 1.67 times (CV: 9.68%) higher than that in the inner shell respectively, as shown in Figure 5d. Although majority has single-cored triple structure, dual- or multi-cored triples are also formed at small fraction, as shown in Figure S5 of the Supporting Information. The microcarriers with single core and double shells show sequential dissolution of p(BMA-co-DAMA-co-MMA)-rich domains in acidic condition. The outer shell is rapidly dissolved within 10 s in buffer solution of pH 5, as shown in Figure 5e and Movie S5 of the Supporting Information; the dissolution time scale is comparable with core-shell microcarriers in Figure 2. By contrast, the core is slowly dissolved due to partial protection with inner PCL shell; it takes approximately 90 s. This dissolution is relatively fast in comparison with inverted core-shell microcarriers. We attribute this to formation of small p(BMA-co-DAMA-co-MMA)-rich

Figure 5. (a, b) Series of illustration and OM images showing the formation of microcarrier composed of pH-responsive core, biodegradable inner shell, and pH-responsive outer shell from homogeneous chloroform drop containing p(BMA-co-DAMA-co-MMA) and PCL in aqueous phase with pH 10. (c) CLSM images of the double-shelled microcarriers. (d) A set of red (left panel), green (middle panel), and overlaid (right panel) fluorescence CLSM images and intensity profile of red and green fluorescence along the dotted line in overlaid image. (e) A Series of CLSM images showing dissolution of pH-responsive outer shell and core at pH 5.

domains in the inner PCL shell due to incomplete phase separation. Microcarriers with Multicores-in-Matrix. Solvent for dispersed phase influences final structure of microcarriers. When toluene is used as solvent for a combination of p(BMA-co-DAMA-co-MMA) and PLGA, instead of chloroform, the emulsion drops evolve to spherical microcarriers composed of many small PLGA-rich domains embedded in a p(BMA-co-DAMA-co-MMA)-rich matrix in a wide range of pH 8.5-10. In the early stage of phase separation, very small PLGA-rich domains are formed in p(BMA-co-DAMA-co-MMA)-rich surrounding in the opposite manner to all the cases with chloroform solvent. The drops do not undergo dramatic coalescence of the small domains and phase inversion during last of consolidation, ending up with pH-responsive microcarriers with small biodegradable particles embedded, as shown in Figure 6a-c and the inset of Figure 6c. Both red and green fluorescence are observed from whole volume of microcarriers in Figure 6d. We attribute this freezing of structure without significant coalescence to fast evaporation of toluene from drops. Toluene drops float immediately underneath the interface between air-continuous phases until toluene is almost depleted due to low density of



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Figure 6. (a, b) Series of illustration and OM images showing the formation of microcarrier composed of biodegradable multicores embedded in pH-responsive matrix from homogeneous toluene drop containing p(BMA-co-DAMA-co-MMA) and PLGA in aqueous phase with pH 8.5; the same structures are obtained in pH 8.5-10. (c) CLSM images of the microcarriers with structure of multicores-in-matrix. (d) A set of red (left panel), green (middle panel), and overlaid (right panel) fluorescence CLSM images and intensity profile of red and green fluorescence along the dotted line in overlaid image. Both red and green fluorescence is observed along the whole volume of microcarriers. (e) A Series of CLSM images showing selective dissolution of pH-responsive core and subsequent release of biodegradable cores at pH 5.

toluene, 0.87 g/ml. Therefore, toluene rapidly vaporizes to air through only the thin layer of water. The consolidation of toluene drops is approximately 2.4-fold faster than that of chloroform drops which sediment on the bottom of glass dish. When the composite microcarriers are subjected to acidic condition of pH 5, p(BMA-co-DAMA-coMMA)-rich matrix is isotropically dissolved in 50 s, thereby releasing Coumarin as shown in Figure 6e and Movie S6 of the Supporting Information. The dissolution leaves behind small PLGA-rich particles, which slowly spread in the continuous phase while retaining Nile red. The PLGArich particles are much smaller than spherical or crescentmoon-shaped compartments left behind from the coreshell or acorn-shaped microcarriers, potentially providing faster release of drugs in PLGA-rich domains. To roughly estimate increase of surface area, we collect and observe the PLGA-rich particles with SEM as shown in Figure S6 of the Supporting Information. Average diameter of the particles is 2.6 μm (CV: 23.0%). Surface area of the particles is approximately 13 times increased in a comparison with single cored particle. Therefore, the microcarriers with multicores-in-matrix will provide faster release of drugs from the multicores. When

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Figure 7. Summary of microcarrier configurations obtained from 12 different sets of biodegradable polymer, pH of continuous phase, and organic solvent.

toluene is used for a combination of p(BMA-co-DAMAco-MMA) and PCL, drops transform to microcarriers with biodegradable core and pH-responsive shell in pH range of 8.5-10. A combination of p(BMA-co-DAMA-co-MMA) and PLA in toluene produces microcarriers with parallel linkage of two domains in pH range of 8.5-10. Only difference of configuration between toluene and chloroform for this combination of polymers is shape of boundary between the two domains; chloroform results in concave boundary for PLA-rich domain, whereas toluene gives relatively flat boundary. All representative configurations of microcarriers obtained by sets of polymers, organic solvents, and pH values are summarized in Figure 7. Release of Model Drugs in Acidic Condition. To study release behaviors of model compounds, we measure time series of fluorescence spectra from microcarriers under acidic conditions. For this, the microcarriers are dried on glass dish, which are then filled with a large amount of pH buffer solution. The microcarriers release dyes to the surrounding and lose their fluorescence over time; the released dyes are highly diluted, which has negligible influence on the spectra. For example, core-shell microcarriers show fluorescence spectrum with two peaks at 510 nm and 600 nm which are contributed from Coumarin and Nile red, respectively, as shown in Figure S7 of


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Chemistry of Materials into the surrounding as matrix is degraded, as we confirmed in Figure 6e; Nile red is still in the small PLGA-rich particles. We expect that Nile red will be released at reduced time scale in multicores-in-matrix in comparison with simple core-shell due to large surface area, as we discussed above.

Figure 8. a, b) Time dependence of normalized fluorescence intensities at wavelength of 510 nm (a) and 600 nm (b) measured from core-shell microcarriers which are under denoted pH values of 7, 6.5, and 6, where emission at 510 nm is predominantly contributed from Coumarin and that at 600 nm is contributed from Nile red. c, d) Time dependence of normalized fluorescence intensities at wavelength of 510 nm (c) and 600 nm (d) measured from the microcarriers with core-shell, inverted core-shell, and multicores-in-matrix configurations, where pH of surrounding is set to be 6.5.

the Supporting Information. The peak intensities decrease over time when the core-shell microcarriers are subjected to acidic condition. To quantitatively compare the release of Coumarin and Nile red from microcarriers, peak intensities at 510 nm and 600 nm are separately plotted over time for three different pH values of 7, 6.5 and 6, as shown in Figure 8a, b. More than 90% Coumarin is released within 2000 s for all three pH values, whereas only half of Nile red is released. This is because the Coumarinconcentrated p(BMA-co-DAMA-co-MMA)-rich compartments are selectively dissolved at the acidic condition. Large reduction of intensity at 600 nm indicates that Nile red is also included in p(BMA-co-DAMA-co-MMA)-rich compartments. Release of the model compounds is faster in lower pH. It takes 450 s to release 80% of encapsulated Coumarin at pH 6, whereas 1410 s at pH 7. We study release profile from three different geometries of microcarriers: core-shell, inverted core-shell, and multicores-in-matrix are used; acorn-shaped and doubleshelled microcarriers show almost same release behaviors to core-shell. The microcarriers are dried and immersed in a buffer solution with pH 6.5. Core-shell microcarriers release more than 90% Coumarin and half of Nile red, as we observed in Figure 8a,b. It takes 690 s to release 80% of encapsulated Coumarin. By contrast, the inverted coreshell microcarriers retain their fluorescence spectra for 1600 s without reduction, indicating negligible release; this is consistent with CLSM images in Figure 4e. Microcarriers with multicores-in-matrix show release of Coumarin in the time scale similar to that of core-shell. At the same time, fluorescence intensity at 600 nm is also decreased. This is because small PLGA-rich particles spread

Selective Release in a Cancer Cells Medium. In general, cancer cells make acidic environment due to the metabolic production of lactic acid by active anaerobic glycolysis.42 Therefore, pH value of the tumor environment is related with the activity of the cancer cells and is used as one of indicators for malignancy of tumors. The microcarriers we designed are able to selectively release therapeutics at acidic environment, thereby being potentially useful for cancer treatment; although vehicles smaller than 200 nm are required to deliver drugs to tumor sites through enhanced permeation and retention (EPR) effect, we believe that our microcarriers can be directly injected into tumor sites or implanted after surgery to release drugs. To investigate release behavior for microcarriers during cultivation of cancer cells, the dispersion of microcarriers is incubated with attached MDAMB-231 breast cancer cells for 24 h. During the incubation, pH of the medium slowly decreases to 6.72. The p(BMAco-DAMA-co-MMA)-rich shells are gently swollen at such a weakly acidic environment, instead of being rapidly dissolved. The shell volume becomes increased almost 5-fold in 24 h, whereas the core remains unchanged, as shown in Figure 9 and Movie S7 of the Supporting Information. This swelling leads to formation of large pores in the shell, allowing slow diffusion of Coumarin to surrounding. By contrast, the core retains Nile red. For a comparison, we incubate acorn-shaped microcarriers in the solution with constant pH of 6.72 and monitor them, as shown in Movie S8. The p(BMA-co-DAMA-co-MMA)-rich compartments are selectively swollen in 40 minutes and dissolved in 1 hour. This experiment indicates that the cultivation medium of cancer cells has higher pH than 6.72 during most period of incubation. We attribute this very slow reduction of pH to ionization of sodium bicarbonate included in the medium; the ionization leads to the production of bicarbonate which suppresses the ionization of carbonic acid.43

Figure 9. A series of fluorescence microscope images of the core-shell microcarriers incubated in MDA-MB-231 cell culture medium.



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CONCLUSION We report a microfluidic approach to create monodisperse microcarriers composed of biodegradable and pHresponsive compartments using phase separation of two immiscible polymers confined in emulsion drops. The emulsion drops containing two polymers are homogeneous at the beginning, which then transform to microcarriers with multiple compartments as solvent is depleted in the drops. Two different model drugs of dye molecules dissolved in the emulsion drops are spontaneously concentrated into their own compartments with higher molecular affinity. Configuration of microcarriers is determined by three control factors: pair of polymers, pH value of continuous phase, and organic solvent. We can exclusively produce uniform microcarriers with unique configuration by controlling these factors from five distinct structures that are core-shell, acorn-shaped, inverted core-shell, core-double shells, and multicores-in-matrix structures. Microcarriers with each configuration provide their own release profile, which potentially enables us to program the release profile of multiple drugs. In particular, as pH-responsive compartments can be dissolved in the weakly acidic environment of cancers, while biodegradable compartment is slowly degraded, the microcarriers can be designed to release two distinct cancer drugs in sequence for combinatorial chemotherapy.44 Moreover, the configuration of microcarriers and therefore release behavior can be further diversified by employing three or more different polymers in the single emulsion drops. Therefore, we believe that this general microfluidic technology will provide new opportunity for designing microcarriers with programmed release function of multiple drugs.

EXPERIMENTAL SECTION Materials. Cationic copolymer, p(BMA-co-DAMA-coMMA) with 1:2:1 molar ratio (Eudragit® EPO, Mw 47,000, Evonik), is used for pH-responsive polymer. PLGA (Mw 113,000, Evonik), PCL (Mw 45,000, Sigma-Aldrich), and PLA (Mw 25,000, Polyscience, Inc.) are used for biodegradable polymers. The cationic copolymer and one of biodegradable polymers are dissolved in either toluene (Sigma-Aldrich) or chloroform (Sigma-Aldrich) to have concentration of 1 w/w% for each polymer; this organic solution is used as a dispersed phase of emulsions. Fluorescent dyes of Nile red (Sigma-Aldrich) and Coumarin (Sigma-Aldrich) are also dissolved in the organic solution with concentrations of 0.05 w/w% and 0.1 w/w% respectively as model drugs. As a continuous phase, aqueous solution of 10 w/w% PVA (Mw 13,000 - 23,000, SigmaAldrich) containing 0.1 M sodium hydroxide (NaOH, Sigma-Aldrich) is used. Device Preparation and Operation. Two cylindrical capillaries (1B100F-6, World precision instruments, Inc.) are tapered by micropipette puller (P97, Sutter Instrument), one of which is then sanded to have larger orifice diameter. These two capillaries are coaxially assembled in

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a square capillary (OD 1.5 mm, ID 1.05 mm, Harvard borosilicate square tubing) without chemical modification. Volumetric flow rates are controlled by syringe pumps (Legato 100, KD Scientific). The flow rates of the dispersed and continuous phases are typically set to be 200 μLh-1 and 1500 μLh-1 and further controlled to maintain dripping mode of drop formation for different sets of fluids and devices. The production of emulsion drops is observed with inverted optical microscope (Eclipse TS100, Nikon) equipped with a high speed camera (Phantom Miro eX2, Vision Research). Drops are collected in the aqueous solution same to the continuous phase at room temperature. The solution is in a wide glass petri dish to have a height of 250 μm which facilitates evaporation of organic solvent from emulsion drops. To maintain high pH of approximately 10 during consolidation, drops collected first 5 minutes and observed. For consolidation at approximately pH 8.5, drops are collected after 1 h and observed; pH value continuously decreases and we take rough mean values during drop consolidation. Microcarriers are washed three times with a basic solution and then stored in phosphate buffer solution with pH 7.4 (Sigma-Aldrich). Characterization. To observe selective dissolution of p(BMA-co-DAMA-co-MMA)-rich domain at acidic condition, dried microcarriers are immersed in pH 5.0 buffer solution and observed with confocal laser scanning microscope (CLSM, LSM 5 PASCAL, Zeiss). After the dissolution, remaining parts are observed with scanning electron microscope (SEM, S-4800, Hitachi). To further confirm the selective dissolution, core-shell microcarriers are incubated in a culture medium of cancer cells, MDA-MB231. For this, MDA-MB-231 cells are plated on 6-well plate (Nunc) in densities of 2 × 105 cells/well in phenol red free RPMI1640 (GIBCO) medium supplemented with 10% fetal bovine serum and grown for 3 days in a humidified incubator containing 5% CO2 at 37 °C. Prior to live cell imaging, the cells are moved and kept on a heated microscope chamber at 37 °C in 5% CO2-humidified atmosphere. The core-shell microcarriers are pipetted into the cell culture medium and the cells are incubated with the core-shell microcarriers for additional 24 h. The images are captured every 10 minutes by using Automated Microscope System with Adaptive Focus Control (DMI6000B, Leica) and the LAS X software (ver. 1,1,12420,0, Leica), designed for acquisition and processing of fluorescence images (Filter set: FL1; green Ex: 450-490 nm, Em: 500-550 nm, FL2; red, Ex: 565-605 nm, Em: 622-698 nm).

ASSOCIATED CONTENT Supporting Information. This material is available free of charge via the Internet at http://pubs.acs.org. OM images showing monodisperse emulsion drops, inverted core-shell microcarriers with defects on the shell, microcarriers with single-, dual-, triple-, multi-cores and double shells, sets of CLSM and SEM images showing two types of microcarriers before and after dissolution, and


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Chemistry of Materials movie clips showing phase inversion and dissolution of microcarriers.

release from homogeneous PLGA microstructures. J. Control. Release 2010, 146, 201−206. (12) Sansdrap, P.; Moës, A. J. Influence of manufacturing parameters on the size characteristics and the release profiles of nifedipine from poly(DL-lactide-co-glycolide) microspheres. Int. J. Pharm. 1993, 98, 157−164.

AUTHOR INFORMATION Corresponding Author * E-mail: [email protected]

Notes The authors declare no competing financial interest.

ACKNOWLEDGMENT This work was supported by the Midcareer Researcher Program (2014R1A2A2A01005813) and Global Research Laboratory (NRF-2015K1A1A2033054) through the National Research Foundation (NRF), funded by the Ministry of Science, ICT and Future Planning (MSIP).

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Microfluidic Production of Uniform Microcarriers with

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